Project Summary/Abstract: Corneal infection is a common cause of vision loss. The corneal epithelium, a barrier to microbe penetration (traversal), has been shown to express various molecules/structures assumed involved in innate defense. Due to the lack of suitable models, specific factors conferring resistance to microbial traversal of epithelia in vivo have not been determined. In the previously funded period, multiple in vivo and in vitro models and methods were developed to enable epithelial cell traversal by microbes to be studied. Together, the models provide the opportunity to do experiments either in the context of, or without, potentially confounding in vivo factors, as needed to test hypotheses. Novel imaging technologies have also been developed that enable bacteria within the corneal epithelium to be imaged, localized, and quantified in intact eyes without the need for tissue dissection, staining, or any other sample preparation/processing. Preliminary data show that removal of tear fluid and subsequent tissue paper blotting (which allows fluorescein to penetrate) enables bacterial adherence to the mouse cornea in vivo, but adherent bacteria do not traverse through the epithelium. Thus, additional epithelial defenses protect against bacterial traversal beyond the superficial barriers that exclude fluorescein. Other in vivo data show that traversal is enabled by; 1) MyD88 knockout, 2) surfactant protein D (SP-D) knockout, or 3) treatment of the corneal surface with EGTA, a calcium chelator known to disrupt cell-to-cell junctions. Data collected using human cells in vitro confirm MyD88 involvement (siRNA knockdown increases traversal). They also show that defense against traversal is boosted by bacterial challenge, and reduced by siRNA knockdown of any of four antimicrobial peptides. The hypothesis to be tested is that MyD88- dependent defense against P. aeruginosa traversal of the corneal epithelium persisting after superficial injury involves bacterial-induced upregulation of surfactant protein D (SP-D), antimicrobial peptides (AMPs) and junctional integrity via TLR or IL-IR mediated signaling. This will be tested in two aims. Aim 1 is to explore the roles of SP-D, antimicrobial factors, and physical barriers in MyD88-dependent defense against bacterial traversal after adhesion. Aim 2 is to determine the receptors and cell types involved in regulating this defense in the corneal epithelium. The general approach will be to utilize MyD88-dependence and bacterial challenge as tools for narrowing down candidate involved factors. Outcomes to be quantified include bacterial traversal, antimicrobial activity, mRNA and protein expression, and colocalization of proteins, bacteria and latex beads of various sizes.